Multi-Arm Block Copolymers as Drug Delivery Vehicles

ABSTRACT

The invention provides methods for making copolymers and multi-arm block copolymers useful as drug delivery vehicles. The multi-arm block copolymers comprise a central core molecule, such as a residue of a polyol, and at least three copolymer arms covalently attached to the central core molecule, each copolymer arm comprising an inner hydrophobic polymer segment covalently attached to the central core molecule and an outer hydrophilic polymer segment covalently attached to the hydrophobic polymer segment, wherein the central core molecule and the hydrophobic polymer segment define a hydrophobic core region. The solubility of hydrophobic biologically active agents can be improved by entrapment within the hydrophobic core region of the block copolymer. The invention further includes pharmaceutical compositions including such block copolymers, pharmaceutical compositions, and methods of using the block copolymers as drug delivery vehicles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/538,013, filed Aug. 7, 2009, which is a continuation of U.S.application Ser. No. 11/894,445, filed Aug. 21, 2007, now U.S. Pat. No.7,589,157, which is a continuation of U.S. application Ser. No.10/984,679, filed Nov. 8, 2004, now U.S. Pat. No. 7,265,186, which is acontinuation-in-part of U.S. application Ser. No. 10/795,913, filed Mar.8, 2004, now U.S. Pat. No. 6,838,528, which is a divisional of U.S.application Ser. No. 10/054,662, filed Jan. 22, 2002, now U.S. Pat. No.6,730,334, which claims the benefit of U.S. Provisional Application Ser.No. 60/262,754, filed Jan. 19, 2001, the disclosures of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to multi-arm copolymers containing a hydrophobiccore region and a hydrophilic outer region, methods of making suchcopolymers, and methods of using such copolymers as drug deliveryvehicles.

BACKGROUND OF THE INVENTION

Solubilization and delivery of hydrophobic drugs is one of the mostchallenging issues in pharmaceutical formulation, particularly sincemost drugs are hydrophobic. Such drugs tend to precipitate in an aqueousenvironment, such as the bloodstream. Whether the drug is delivered byoral or parenteral routes, a certain level of aqueous solubility isrequired for adequate absorption and bioavailability. Pharmaceuticalgrade surfactants, such as Tween® 80 or Cremophor®, have been widelyused in formulations to compensate for the low aqueous solubility ofhydrophobic drugs. These surfactants solubilize hydrophobic drugs byforming micellar structures in aqueous media. Unfortunately, thesesurfactants have been associated with severe allergic reactions andhypersensitivity when administered to patients (Kris, et al., CancerTreatment REP, 70:5, (1986)). After parenteral administration, thesemicellar drug carriers disintegrate when the concentration is belowtheir critical micelle concentration (CMC), resulting in a rapid releaseof the drug. That is to say, in addition to the possibility of adverseside effects upon administration, conventional surfactant-based carriersalso lack the ability to provide controlled release of a drug.

Thus, there remains a need in the art for a method for impartingadequate levels of aqueous solubility to a hydrophobic drug such thatthe drug may be administered in a therapeutically effective manner.

SUMMARY OF THE INVENTION

The invention is directed to multi-arm block copolymers useful as drugdelivery vehicles. The multi-arm block copolymers comprise a centralcore molecule, such as a residue of a polyol, and at least threecopolymer arms covalently attached to the central core molecule, eachcopolymer arm comprising an inner hydrophobic polymer segment covalentlyattached to the central core molecule and an outer hydrophilic polymersegment covalently attached to the hydrophobic polymer segment. Theblock copolymer provides a unimolecular micelle structure, wherein thecentral core molecule and the hydrophobic polymer segment define ahydrophobic core region and the hydrophilic polymer segment defines anouter hydrophilic region. The solubility of hydrophobic biologicallyactive agents can be improved by entrapment within the hydrophobic coreregion of the block copolymer. Thus, improved delivery of hydrophobicdrugs can be obtained by administering a pharmaceutical composition to amammal, the pharmaceutical composition comprising a multi-arm blockcopolymer of the invention having a drug entrapped within thehydrophobic core region thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying figures, wherein.

FIG. 1 is an illustration of the structure of an embodiment of themulti-arm block copolymer of the invention;

FIG. 2 provides release profiles for the drug, 3,4-di-[1-methyl6-nitro-3-indolyl]-1H-pyrrole-2,5-dione (MNIPD), in several polymercompositions;

FIG. 3 provides release profiles for the drug, simvastatin, in severalpolymer compositions;

FIG. 4 provides a release profile for simvastatin in an exemplarybisphosphonate derivative of a multi-arm block copolymer;

FIG. 5 provides release profiles for the drug, paclitaxel, in twomulti-arm block copolymer embodiments of the invention;

FIG. 6 provides release profiles for the drug, indomethacin, in severalpolymer compositions;

FIG. 7 provides release profiles for the drug, pivaloxymethyl butyrate,in two multi-arm block copolymer embodiments of the invention;

FIG. 8 provides a release profile for the drug, cyclosporin A, in amulti-arm block copolymer embodiment of the invention;

FIG. 9 provides a release profile for the drug, paclitaxel, in amulti-arm block copolymer embodiment of the invention;

FIG. 10 provides a comparison of the in vivo effect of a conventionalTaxol® formulation versus an 8-arm poly(lactide)-mPEG blockcopolymer/Taxol® formulation of the invention on lung tumor growth; and

FIG. 11 is an example of Dynamic Light Scattering (DLS) data.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

I. Definitions

The terms “functional group”, “active moiety”, “activating group”,“reactive site”, “chemically reactive group” and “chemically reactivemoiety” are used in the art and herein to refer to distinct, definableportions or units of a molecule. The terms are somewhat synonymous inthe chemical arts and are used herein to indicate the portions ofmolecules that perform some function or activity and are reactive withother molecules. The term “active,” when used in conjunction withfunctional groups, is intended to include those functional groups thatreact readily with electrophilic or nucleophilic groups on othermolecules, in contrast to those groups that require strong catalysts orhighly impractical reaction conditions in order to react (i.e.,“non-reactive” or “inert” groups). For example, as would be understoodin the art, the term “active ester” would include those esters thatreact readily with nucleophilic groups such as amines. Exemplary activeesters include N-hydroxysuccinimidyl esters or 1-benzotriazolyl esters.Typically, an active ester will react with an amine in aqueous medium ina matter of minutes, whereas certain esters, such as methyl or ethylesters, require a strong catalyst in order to react with a nucleophilicgroup.

The term “linkage” or “linker” is used herein to refer to an atom,groups of atoms, or bonds that are normally formed as the result of achemical reaction. A linker of the invention typically links theconnecting moieties, such two polymer segments, via one or more covalentbonds. Hydrolytically stable linkages means that the linkages aresubstantially stable in water and do not react to any significant degreewith water at useful pHs, e.g., under physiological conditions for anextended period of time, perhaps even indefinitely. Hydrolyticallyunstable or degradable linkages means that the linkages are degradablein water or in aqueous solutions, including for example, blood.Enzymatically unstable or degradable linkages means that the linkage canbe degraded by one or more enzymes.

The term “alkyl” refers to hydrocarbon chains typically ranging fromabout 1 to about 12 carbon atoms in length, and includes straight andbranched chains. The hydrocarbon chains may be saturated or unsaturated.The term “substituted alkyl” refers to an alkyl group substituted withone or more non-interfering substituents, such as, but not limited to,C3-C6 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like;acetylene; cyano; alkoxy, e.g., methoxy, ethoxy, and the like; loweralkanoyloxy, e.g., acetoxy; hydroxy; carboxyl; amino; lower alkylamino,e.g., methylamino; ketone; halo, e.g. chloro or bromo; phenyl;substituted phenyl, and the like.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C1-C6 alkyl (e.g., methoxy or ethoxy).

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Multiple aryl rings may be fused, as in naphthyl or unfused, asin biphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Substituted aryl” is aryl having one or more non-interfering groups assubstituents. For substitutions on a phenyl ring, the substituents maybe in any orientation (i.e., ortho, meta or para).

“Heteroaryl” is an aryl group containing from one to four N, O, or Satoms(s) or a combination thereof, which heteroaryl group is optionallysubstituted at carbon or nitrogen atom(s) with C1-6 alkyl, —CF₃, phenyl,benzyl, or thienyl, or a carbon atom in the heteroaryl group togetherwith an oxygen atom form a carbonyl group, or which heteroaryl group isoptionally fused with a phenyl ring. Heteroaryl rings may also be fusedwith one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroarylrings. Heteroaryl includes, but is not limited to, 5-memberedheteroaryls having one hetero atom (e.g., thiophenes, pyrroles, furans);5 membered heteroaryls having two heteroatoms in 1, 2 or 1,3 positions(e.g., oxazoles, pyrazoles, imidazoles, thiazoles, purines); 5-memberedheteroaryls having three heteroatoms (e.g., triazoles, thiadiazoles);5-membered heteroaryls having 3 heteroatoms; 6-membered heteroaryls withone heteroatom (e.g., pyridine, quinoline, isoquinoline, phenanthrine,5,6-cycloheptenopyridine); 6-membered heteroaryls with two heteroatoms(e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines,quinazolines); 6-membered heretoaryls with three heteroatoms (e.g.,1,3,5-triazine); and 6-membered heteroaryls with four heteroatoms.

“Substituted heteroaryl” is heteroaryl having one or morenon—interfering groups as substituents.

“Heterocycle” or “heterocyclic” means one or more rings of 5, 6 or 7atoms with or without unsaturation or aromatic character and at leastone ring atom which is not carbon. Preferred heteroatoms include sulfur,oxygen, and nitrogen. Multiple rings may be fused, as in quinoline orbenzofuran.

“Substituted heterocycle” is heterocycle having one or more side chainsformed from non-interfering substituents.

“Non-interfering substituents” are those groups that yield stablecompounds. Suitable non-interfering substituents or radicals include,but are not limited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10alkynyl, C1-C10 alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C3-C10cycloalkyl, C3-C10 cycloalkenyl, phenyl, substituted phenyl, toluoyl,xylenyl, biphenyl, C2-C12 alkoxyalkyl, C7-C12 alkoxyaryl, C7-C12aryloxyalkyl, C6-C12 oxyaryl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl,—(CH₂)_(m)—O—(C1-C10 alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NRC(O)—(C1-C10 alkyl),—C(O)—(C1-C10 alkyl), C2-C10 thioalkyl, —C(O)O—(C1-C10 alkyl), —OH,—SO₂, ═S, —COOH, —NR, carbonyl, —C(O)—(C1-C10 alkyl)-CF₃, —C(O)—CF₃,—C(O)NR₂, —(C1-C10 alkyl)-S—(C6-C12 aryl), —C(O)—(C6-C12 aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C1-C10 alkyl) wherein each m is from 1 to 8,—C(O)NR, —C(S)NR, —SO₂NR, —NRC(O)NR, —NRC(S)NR, salts thereof, and thelike. Each R as used herein is H, alkyl or substituted alkyl, aryl orsubstituted aryl, aralkyl, or alkaryl.

The term “drug”, “biologically active molecule”, “biologically activemoiety” or “biologically active agent”, when used herein means anysubstance which can affect any physical or biochemical properties of abiological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular, as used herein,biologically active molecules include any substance intended fordiagnosis, cure mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs, dyes, lipids, nucleosides, oligonucleotides,cells, viruses, liposomes, microparticles and micelles. Classes ofbiologically active agents that are suitable for use with the inventioninclude, but are not limited to, antibiotics, fungicides, anti-viralagents, anti-inflammatory agents, anti-tumor agents, cardiovascularagents, anti-anxiety agents, hormones, growth factors, steroidal agents,and the like.

“Hydrophobic” refers to molecules having a greater solubility in octanolthan in water, typically having a much greater solubility in octanol.Conversely, “hydrophilic” refers to molecules having a greatersolubility in water than in octanol.

“Poly(hydroxyester)” refers to polymers comprising repeating monomerunits of —O—R—C(O)—, wherein R is alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, orsubstituted heterocycle. Exemplary poly(hydroxyesters) includepoly(lactide), poly(glycolide), poly(lactide/glycolide) copolymer,poly(butyrolactide), and polycaprolactone.

“Oligomer” refers to short monomer chains comprising 2 to about 10monomer units.

II. The Multi-Arm Block Copolymer

In one aspect, the present invention provides a multi-arm blockcopolymer having a hydrophobic core region defined by a central coremolecule and hydrophobic polymer arms covalently attached to the centralcore molecule and an outer hydrophilic region defined by a hydrophilicpolymer covalently attached to the hydrophobic polymer arms. Each arm ofthe multi-arm structure comprises an inner (i.e. closer to the centralcore molecule) hydrophobic polymer segment and an outer (i.e. furtherfrom the central core molecule) hydrophilic polymer segment.

In aqueous solution, it is believed that the multi-arm block copolymeracts as a unimolecular micelle having a central hydrophobic core regionbounded by a hydrophilic region. As demonstrated in the experimentalsection, the multi-arm block copolymers of the invention are capable ofincreasing the aqueous solubility of hydrophobic biologically activeagents or drugs by encapsulating or physically entrapping thehydrophobic drug molecule within the hydrophobic core region of themulti-arm block copolymer structure. Thus, the multi-arm blockcopolymers are useful as drug delivery vehicles, particularly forhydrophobic drug molecules. “Encapsulation” or “entrapment” is intendedto refer to physical confinement of the drug molecule within thehydrophobic region of the copolymer, rather than covalent attachment tothe copolymer.

Compared to conventional linear micelle structures, the unimolecularnature of the multi-arm block copolymers of the invention results inless sensitivity to concentration, such that the block copolymers of theinvention are less likely to release the entrapped drug molecules at anundesirably rapid rate. The multi-arm block copolymers of the inventionare covalently bound molecular units rather than molecular aggregatesand, thus, are substantially precluded from disassembly in circulationin the absence of hydrolytically unstable linkages within the polymersegments specifically intended to degrade the copolymer. Further, sincechemical modification of the drug molecules is not required to obtain anincrease in solubility, the possibility of the copolymer reducingefficacy of the entrapped drug is greatly reduced.

Although not bound by any particular theory, it is believed that thelevel of hydrophobicity and size of the hydrophobic polymer affect thedrug loading and drug release characteristics of the multi-arm blockcopolymer. In general, it is believed that larger hydrophobic polymersegments and hydrophobic polymer segments formed from polymers havingrelatively greater degrees of hydrophobicity will result in higher drugloading and slower drug release profiles in solution. Conversely,smaller hydrophobic polymer segments and hydrophobic polymer segmentsformed from polymers having relatively lower degrees of hydrophobicitywill result in reduced drug loading and more rapid drug release.

Further, without being bound by theory, it is believed that the numberof arms of the multi-arm block polymer also impacts the drug loading anddrug release characteristics of the copolymer. Generally, the presenceof fewer copolymer arms results in reduced drug loading. However, theuse of a copolymer with a very large number of arms can also reduce drugloading because of the substantial increase in density and concomitantreduction in interstitial space within the hydrophobic core region ofthe copolymer structure. Generally, the presence of fewer copolymer armswill also result in more rapid drug release. This is attributed, atleast in part, to the effect of aggregation of multi-arm blockcopolymers and entrapment of drug molecules within a hydrophobic regiondefined by the aggregated copolymers. Aggregation of the multi-arm blockcopolymers creates hydrophobic regions that are not unimolecular innature. Instead, a multi-arm block copolymer aggregate behaves in amanner analogous to conventional linear micelles. Reductions inconcentration can break up the copolymer aggregate and release a portionof the drug molecules entrapped within the hydrophobic region created bythe aggregation. Copolymers with a higher number of arms are lesssusceptible to the aggregation effect and less likely to have drugrelease characteristics that depend on concentration. In light of theforegoing, an optimal range for the number of arms of the blockcopolymer can be determined such that both desirable drug loading anddrug release characteristics are obtained for any particular hydrophobicdrug. In most embodiments, the number of arms is in the range of 3 toabout 25, preferably at least 5, more preferably at least about 8, andmost preferably at least about 10.

The hydrophobic and hydrophilic polymer segments are preferably not“hyper-branched” or dendritic in nature, such as the dendrimersdescribed in U.S. Pat. No. 5,830,986, wherein branched compounds areattached in numerous successive layers to a central core. Instead, bothpolymer segments are preferably substantially linear in nature asdepicted in FIG. 1. However, some branching in either polymer segmentmay be present. For example, a branched poly(ethylene glycol) polymercomprising two polymer backbones attached to lysine linker is used asthe hydrophilic polymer in several appended examples.

Although the specific examples of multi-arm block copolymers in theappended experimental section utilize the same block copolymer structurefor each copolymer arm, it is possible to utilize different copolymerstructures within the same multi-arm structure. In other words, thepresent invention includes embodiments wherein more than one particularhydrophobic/hydrophilic polymer combination is attached to the same coremolecule.

A. The Central Core

The central core molecule is derived from a molecule that provides anumber of polymer attachment sites equal to the number of desiredcopolymer arms. Preferably, the central core molecule of the multi-armblock copolymer structure is the residue of a polyol having at leastthree hydroxyl groups available for polymer attachment. A “polyol” is amolecule comprising a plurality of available hydroxyl groups. Dependingon the desired number of copolymer arms, the polyol will typicallycomprise 3 to about 25 hydroxyl groups, preferably at least 5, morepreferably at least about 8, and most preferably at least about 10. Thepolyol may include other protected or unprotected functional groups aswell without departing from the invention. Although the spacing betweenhydroxyl groups will vary from polyol to polyol, there are typically 1to about 20 atoms, such as carbon atoms, between each hydroxyl group,preferably 1 to about 5. As would be understood in the art, by “residue”is meant the portion of the polyol molecule remaining after attachmentof the copolymer arms. Preferred polyols include glycerol, reducingsugars such as sorbitol, pentaerythritol, and glycerol oligomers, suchas hexaglycerol. As noted in the appended examples, a 21-arm blockcopolymer can be synthesized using hydroxypropyl-β-cyclodextrin, whichhas 21 available hydroxyl groups. The particular polyol chosen willdepend on the desired number of hydroxyl groups needed for attachment tothe copolymer arms.

B. The Hydrophobic Polymer

The particular hydrophobic polymer used in the present invention willdepend, at least in part, on the desired drug loading and drug releasecharacteristics, since as explained above, the size and hydrophobicityof the hydrophobic polymer segment will affect those characteristics.The hydrophobic polymer should be generally non-toxic and biocompatible,meaning that the polymer is capable of coexistence with living tissuesor organisms without causing harm. In preferred embodiments, thehydrophobic polymer segments comprises a poly(hydroxyester), apoly(alkylene oxide) other than poly(ethylene glycol), such aspoly(propylene oxide) (PPO) or poly(butylene oxide) (PBO), or copolymersthereof. Exemplary poly(hydroxyester) polymers include poly(lactide),poly(glycolide), poly(lactide/glycolide) copolymer, poly(butyrolactide)and polycaprolactone. The hydrophobic polymer segment of the blockcopolymer will typically have a number average molecular weight of about500 Da to about 100,000 Da, preferably about 10,000 Da to about 40,000Da. For example, hydrophobic polymer segments having a molecular weightof about 5,000 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da,about 25,000 Da or about 30,000 Da are useful in the present invention.

In addition to being hydrophobic, the poly(hydroxyester) polymers alsoinclude one or more hydrolytically or enzymatically degradable linkages,such as ester linkages. Typically, use of these polymers results in theformation of degradable linkages between the central core molecule andthe polymer segment, within the polymer segment, between the hydrophobicpolymer segment and the hydrophilic polymer segment, or some combinationthereof. As used herein, the hydrophobic polymer is said to comprise adegradable linkage if a linkage is located at any of the above-listedlocations. The use of a hydrophobic polymer with one or more degradablelinkages allows the multi-arm block copolymer to degrade in solutionover time, thus increasing renal clearance of the copolymer. Inaddition, the degradable linkages provide an additional feature of thesepolymers, i.e., the ability to control the rate of release of theentrapped drug.

C. The Hydrophilic Polymer

The hydrophilic polymer segment may comprise any hydrophilic polymer. Aswith the hydrophobic polymer, the hydrophilic polymer should begenerally non-toxic and biocompatible, meaning that the polymer iscapable of coexistence with living tissues or organisms without causingharm. Preferably, poly(ethylene glycol) (PEG) is used as the hydrophilicpolymer segment. The term PEG includes poly(ethylene glycol) in any ofits linear, branched or multi-arm forms, including alkoxy PEG,bifunctional PEG, forked PEG, branched PEG, pendant PEG, or PEG withdegradable linkages therein, to be more fully described below.

In its simplest form, PEG has the formula—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about 10 to about 4000,typically from about 20 to about 500. PEGs having a number averagemolecular weight of from about 500 Da to about 100,000 Da, preferablyabout 1,000 Da to about 20,000 Da are particularly useful as thehydrophilic polymer segment. For example, PEG polymer segments having amolecular weight of about 1,000 Da, about 5,000 Da, about 10,000 Da,about 15,000 Da, or about 20,000 Da are useful in the present invention.

In one form useful in the present invention, free or non-bound PEG is alinear polymer terminated at each end with hydroxyl groups:

HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the—PEG-symbol represents the following structural unit:

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—

where n typically ranges from about 10 to about 4000.

Another type of PEG useful in forming the conjugates of the invention ismethoxy-PEG-OH, or mPEG in brief, in which one terminus is therelatively inert methoxy group, while the other terminus is a hydroxylgroup that is subject to ready chemical modification. The structure ofmPEG is given below.

CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

where n is as described above. The use of hydrophilic polymer segmentsin the form of mPEG is exemplified in Examples 1 and 4.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the hydrophilic PEG polymer segment. Forexample, the hydrophilic PEG segment can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones, such as methoxy poly(ethyleneglycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched polymer segment comprises methoxy poly(ethylene glycol)disubstituted lysine. Use of such a branched PEG structure isexemplified in Examples 2, 5, and 7.

The PEG polymer may alternatively comprise a forked PEG. An example of aforked PEG is represented by PEG-YCHZ₂, where Y is a linking group and Zis an activated terminal group linked to CH by a chain of atoms ofdefined length. International Application No. PCT/US99/05333, thecontents of which are incorporated by reference herein, disclosesvarious forked PEG structures capable of use in the present invention.The chain of atoms linking the Z functional groups to the branchingcarbon atom serve as a tethering group and may comprise, for example,alkyl chains, ether chains, ester chains, amide chains and combinationsthereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG segment rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG segment directly or through alinking moiety, such as alkylene.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the segment,including any of the above described polymers. For example, PEG can beprepared with ester linkages in the polymer segment that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

—PEG-CO₂—PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Similarly, the PEG polymer can be covalently attached to the hydrophobicpolymer segment or other molecules through a weak or degradable linkagemoiety.

Other hydrolytically degradable linkages, useful as either a degradablelinkage within a polymer segment or as a degradable linkage connectingthe PEG polymer to other molecules include carbonate linkages; iminelinkages resulting, for example, from reaction of an amine and analdehyde (see, e.g., Ouchi et al., Polymer Preprints, 38(1):582-3(1997), which is incorporated herein by reference.); phosphate esterlinkages formed, for example, by reacting an alcohol with a phosphategroup; hydrazone linkages which are typically formed by reaction of ahydrazide and an aldehyde; acetal linkages that are typically formed byreaction between an aldehyde and an alcohol; orthoester linkages thatare, for example, formed by reaction between a formate and an alcohol;peptide linkages foamed by an amine group, e.g., at an end of a polymersuch as PEG, and a carboxyl group of a peptide; and oligonucleotidelinkages formed by, for example, a phosphoramidite group, e.g., at theend of a polymer, and a 5′ hydroxyl group of an oligonucleotide.

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms of PEG.

In some embodiments, it may be desirable to covalently attach atargeting moiety or drug molecule to the hydrophilic polymer segment. Asused herein, “targeting moiety” includes any chemical moiety capable ofbinding to, or otherwise exhibiting an affinity for, a particular typeof tissue or component thereof. The addition of a targeting moiety tothe copolymer structure can direct the copolymer to particular siteswithin the body for targeted release of the physically entrapped drug.For example, certain moieties are known to exhibit an affinity forhydroxyapatite surfaces (i.e. calcium phosphate), such as bone.Exemplary hydroxyapatite-targeting moieties include tetracycline,calcein, bisphosphonates, such as4-amino-1-hydroxybutane-1,1-diphosphonic acid, ditetrabutylammonium salt(AHBDP) or derivatives thereof, polyaspartic acid, polyglutamic acid,and aminophosphosugars. Additional targeting moieties include proteins,antibodies, antibody fragments, peptides, carbohydrates, lipids,oligonucleotides, DNA, RNA, or small molecules having a molecular weightless than 2000 Daltons.

The PEG polymer segment may further include one or more capping groupscovalently attached to the PEG molecule, such as at a terminus of thePEG segment distal from the point of attachment to the hydrophobicpolymer. The capping group can be a relatively inert group, such as analkoxy group (e.g. methoxy or ethoxy). Alternatively, the capping groupcan be a reactive functional group, such as a functional group capableof reacting with a targeting moiety or drug molecule so that suchmolecules can be attached to the PEG polymer as described above.Exemplary functional groups, optionally in protected form, includehydroxyl, protected hydroxyl, active ester (e.g. N-hydroxysuccinimidyl,1-benzotriazolyl, p-nitrophenyl, or imidazolyl esters), active carbonate(e.g. N-hydroxysuccinimidyl, 1-benzotriazolyl, p-nitrophenyl, orimidazolyl carbonate), acetal, aldehyde, aldehyde hydrates, alkyl oraryl sulfonate, halide, disulfide derivatives such as o-pyridyldisulfidyl, alkenyl, acrylate, methacrylate, acrylamide, active sulfone,amine, protected amine, hydrazide, protected hydrazide, thiol, protectedthiol, carboxylic acid, protected carboxylic acid, isocyanate,isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, ortresylate.

As would be understood in the art, the term “protected” refers to thepresence of a protecting group or moiety that prevents reaction of thechemically reactive functional group under certain reaction conditions.The protecting group will vary depending on the type of chemicallyreactive group being protected and the reaction conditions employed. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the invention, see for example, Greene, T. W., et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, 2nd ed., John Wiley & Sons, New York, N.Y.(1991).

Specific examples of functional groups for the hydrophilic polymerinclude N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zaplipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zaplipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al. Macrolol. Chem.180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141(1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341(1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461). All of the above references are incorporatedherein by reference.

D. Exemplary Multi-Arm Block Copolymer Structures

More specific structural embodiments of the block copolymers of theinvention will now be described. The specific structures shown below arepresented as exemplary structures only, and are not intended to limitthe scope of the invention.

In one embodiment, a block copolymer of the invention is represented byFormula II:

A(—O—B—O—C-D)_(n)

wherein:

-   -   A is a central core molecule as described above, such as a        residue of a polyol having at least three hydroxyl groups,    -   O is oxygen,    -   B is a hydrophobic polymer segment as described above,    -   C is a hydrophilic polymer segment as described above,    -   D is a capping group as described above, and    -   n is 3 to about 25, preferably at least about 5, more preferably        at least about 8, and most preferably at least about 10.

In a further embodiment, the block copolymer has the following structurerepresented by Formula III:

(E-C—O—B—O—)_(p)A(—O—B—O—C-D)_(m)

wherein:

-   -   A, O, B, C are as described above,    -   D is an alkoxy or hydroxy group,    -   p is at least 1,    -   the sum of m and p is from 3 to about 25, and    -   E is a functional group as described above.

In a third embodiment, the copolymer has the following structurerepresented by Formula IV:

(T-C—O—B—O—)_(p)A(—O—B—O—C-D)_(m)

wherein:

-   -   A, O, B, C are as described above,    -   D is a capping group,    -   p is at least 1,    -   the sum of m and p is from 3 to about 25, and    -   T is a targeting moiety or drug moiety as described above, such        as a bisphosphonate.

Regarding Formulas III and IV above, in one embodiment, p is 1 to about5, preferably 1 to about 3, and the sum of m and p is about 6 to about21, preferably about 8 to about 15.

Formula V below is an exemplary 8-arm PPO-PEG block copolymer made inaccordance with the invention:

Formula VI below is an exemplary 8-arm degradablepoly(lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer of theinvention:

E. The Hydrophobic Drug

The hydrophobic biologically active moiety or drug may be anybiologically active hydrophobic compound that would benefit fromincreased aqueous solubility. The entrapped or encapsulated drug may beutilized per se or in the form of a pharmaceutically acceptable salt. Ifused, a salt of the drug compound should be both pharmacologically andpharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare the free active compound orpharmaceutically acceptable salts thereof and are not excluded from thescope of this invention. Such pharmacologically and pharmaceuticallyacceptable salts can be prepared by reaction of the drug with an organicor inorganic acid, using standard methods detailed in the literature.Examples of useful salts include, but are not limited to, those preparedfrom the following acids: hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric,citric, methanesulphonic, formic, malonic, succinic,naphthalene-2-sulphonic and benzenesulphonic, and the like. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium, or calcium salts of acarboxylic acid group.

Examples of hydrophobic drug molecules that may be encapsulated withinthe multi-arm block copolymers of the invention include, but are notlimited to, abietic acid, aceglatone, acenaphthene, acenocoumarol,acetohexamide, acetomeroctol, acetoxolone, acetyldigitoxins, acetylenedibromide, acetylene dichloride, acetylsalicylic acid, alantolactone,aldrin, alexitol sodium, allethrin, allylestrenol, allylsulfide,alprazolam, aluminum bis(acetylsalicylate), ambucetamide,aminochlothenoxazin, aminoglutethimide, amyl. chloride, androstenediol,anethole trithone, anilazine, anthralin, Antimycin A, aplasmomycin,arsenoacetic acid, asiaticoside, astemizole, aurodox, aurothioglycanide,8-azaguanine, azobenzene, baicalein, Balsam Peru, Balsam Tolu, barban,baxtrobin, bendazac, bendazol, bendroflumethiazide, benomyl, benzathine,benzestrol, benzodepa, benzoxiquinone, benzphetamine, benzthiazide,benzyl benzoate, benzyl cinnamate, bibrocathol, bifenox, binapacryl,bioresmethrin, bisabolol, bisacodyl, bis(chlorophenoxy)methane, bismuthiodosubgallate, bismuth subgallate, bismuth tannate, Bisphenol A,bithionol, bornyl, bromoisovalerate, bornyl chloride, bornylisovalerate, bornyl salicylate, brodifacoum, bromethalin,broxyquinoline, bufexamac, butamirate, butethal, buthiobate, butylatedhydroxyanisole, butylated hydroxytoluene, calcium iodostearate, calciumsaccharate, calcium stearate, capobenic acid, captan, carbamazepine,carbocloral, carbophenothin, carboquone, carotene, carvacrol,cephaeline, cephalin, chaulmoogric acid, chenodiol, chitin, chlordane,chlorfenac, chlorfenethol, chlorothalonil, chlorotrianisene,chlorprothixene, chlorquinaldol, chromonar, cilostazol, cinchonidine,citral, clinofibrate, clofaziminc, clofibrate, cloflucarban, clonitrate,clopidol, clorindione, cloxazolam, coroxon, corticosterone, cournachlor,coumaphos, coumithoate cresyl acetate, crimidine, crufomate, cuprobam,cyamemazine, cyclandelate, cyclarbamate cymarin, cyclosporin A,cypermethril, dapsone, defosfamide, deltamethrin, deoxycorticocosteroneacetate, desoximetasone, dextromoramide, diacetazoto, dialifor,diathymosulfone, decapthon, dichlofluani, dichlorophen,dichlorphenamide, dicofol, dicryl, dicumarol, dienestrol,diethylstilbestrol, difenamizole, dihydrocodeinone enol acetate,dihydroergotamine, dihydromorphine, dihydrotachysterol, dimestrol,dimethisterone, dioxathion, diphenane,N-(1,2-diphenylethyl)nicotinamide, 3,4-di-[1-methyl6-nitro-3-indolyl]-1H-pyrrole-2,5-dione (MNIPD), dipyrocetyl,disulfamide, dithianone, doxenitoin, drazoxolon, durapatite, edifenphos,emodin, enfenamic acid, erbon, ergocorninine, erythrityl tetranitrate,erythromycin stearate, estriol, ethaverine, ethisterone, ethylbiscournacetate, ethylhydrocupreine, ethyl menthane carboxamide,eugenol, euprocin, exalamide, febarbamate, fenalamide, fenbendazole,fenipentol, fenitrothion, fenofibrate, fenquizone, fenthion, feprazone,flilpin, filixic acid, floctafenine, fluanisone, flumequine, fluocortinbutyl, fluoxymesterone, fluorothyl, flutazolam, fumagillin,5-furftiryl-5-isopropylbarbituric acid, fusaftmgine; glafenine,glucagon, glutethimide, glybuthiazole, griseofulvin, guaiacol carbonate,guaiacol phosphate; halcinonide, hematoporphyrin, hexachlorophene,hexestrol, hexetidine, hexobarbital, hydrochlorothiazide, hydrocodone,ibuproxam, idebenone, indomethacin, inositol niacinate, iobenzamic acid,iocetamic acid, iodipamide, iomeglamic acid, ipodate, isometheptene,isonoxin, 2-isovalerylindane-1,3-dione, josamycin, 11-ketoprogesterone,laurocapram, 3-O-lauroylpyridoxol diacetate, lidocaine, lindane,linolenic acid, liothyronine, lucensomycin, mancozeb, mandelic acid,isoamyl ester, mazindol, mebendazole, mebhydroline, mebiquine,melarsoprol, melphalan, menadione, menthyl valerate, mephenoxalone,mephentermine, mephenyloin, meprylcaine, mestanolone, mestranol,mesulfen, metergoline, methallatal, methandriol, methaqualone,methylcholanthrene, methylphenidate, 17-methyltestosterone,metipranolol, minaprine, myoral, naftalofos, naftopidil, naphthalene,2-naphthyl lactate, 2-(2-naphthyloxy)ethanol, naphthyl salicylate,naproxen, nealbarbital, nemadectin, niclosamide, nicoclonate,nicomorphine, nifuroquine, nifuroxazide, nitracrine, nitromersol,nogalamycin, nordazepam, norethandrolone, norgestrienone, octaverine,oleandrin, oleic acid, oxazepam, oxazolam, oxeladin, oxwthazaine,oxycodone, oxymesterone, oxyphenistan acetate, paclitaxel,paraherquamide, parathion, pemoline, pentaerythritol tetranitrate,pentylphenol, perphenazine, phencarbamide, pheniramine,2-phenyl-6-chlorophenol, phenthnethylbarbituric acid, phenyloin,phosalone, O-phthalylsulfathiazole, phylloquinone, picadex, pifamine,piketopfen, piprozolin, pirozadil, pivaloyloxymethyl butyrate,plafibride, plaunotol, polaprezinc, polythiazide, probenecid,progesterone, promegestone, propanidid, propargite, propham, proquazone,protionamide, pyrimethamine, pyrimithate, pyrvinium pamoate, quercetin,quinbolone, quizalofo-ethyl, rafoxanide, rescinnamine, rociverine,ronnel, salen, scarlet red, siccanin, simazine, simetride, simvastatin,sobuzoxane, solan, spironolactone, squalene, stanolone, sucralfate,sulfabenz, sulfaguanole, sulfasalazine, sulfoxide, sulpiride,suxibuzone, talbutal, terguide, testosterone, tetrabromocresol,tetrandrine, thiacetazone, thiocolchicine, thioctic acid, thioquinox,thioridazine, thiram, thymyl N-isoamylcarbamate, tioxidazole, tioxolone,tocopherol, tolciclate, tolnaftate, triclosan, triflusal, triparanol,ursolic acid, valinomycin, verapamil, vinblastine, vitamin A, vitamin D,vitamin E, xenbucin, xylazine, zaltoprofen, and zearalenone.

III. Pharmaceutical Compositions Comprising the Multi-Arm BlockCopolymer

In another aspect, the invention provides pharmaceutical formulations orcompositions, both for veterinary and for human medical use, comprisinga multi-arm block copolymer as described above and at least onebiologically active agent entrapped within the hydrophobic core regionof the multi-arm block copolymer. As noted previously, incorporation ofa hydrophobic drug into the block copolymer structure of the inventionincreases the aqueous solubility of the drug, which can enhance thecirculating residence time of the drug upon administration to a mammal.

The pharmaceutical formulation may include one or more pharmaceuticallyacceptable carriers, and optionally any other therapeutic ingredients,stabilizers, or the like. The carrier(s) must be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not unduly deleterious to the recipient thereof.The compositions of the invention may also include polymericexcipients/additives or carriers, e.g., polyvinylpyrrolidones,derivatized celluloses such as hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (apolymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin andsulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin. Thecompositions may further include diluents, buffers, binders,disintegrants, thickeners, lubricants, preservatives (includingantioxidants), flavoring agents, taste-masking agents, inorganic salts(e.g., sodium chloride), antimicrobial agents (e.g., benzalkoniumchloride), sweeteners, antistatic agents, surfactants (e.g.,polysorbates such as “TWEEN 20” and “TWEEN 80”, and pluronics such asF68 and F88, available from BASF), sorbitan esters, lipids (e.g.,phospholipids such as lecithin and other phosphatidylcholines,phosphatidylethanolamines, fatty acids and fatty esters, steroids (e.g.,cholesterol)), and chelating agents (e.g., EDTA, zinc and other suchsuitable cations). Other pharmaceutical excipients and/or additivessuitable for use in the compositions according to the invention arelisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and in “Handbookof Pharmaceutical Excipients”, Third Ed., Ed. A. H. Kibbe,Pharmaceutical Press, 2000.

The block copolymers of the invention may be formulated in compositionsincluding those suitable for oral, buccal, rectal, topical, nasal,ophthalmic, or parenteral (including intraperitoneal, intravenous,subcutaneous, or intramuscular injection) administration. The blockcopolymers may also be used in formulations suitable for inhalation. Thecompositions may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy. Allmethods include the step of bringing the block copolymer with drugentrapped therein into association with a carrier that constitutes oneor more accessory ingredients. In general, the compositions are preparedby bringing the block copolymer/drug formulation into association with aliquid carrier to form a solution or a suspension, or alternatively,bringing the block copolymer/drug formulation into association withformulation components suitable for forming a solid, optionally aparticulate product, and then, if warranted, shaping the product into adesired delivery form. Solid formulations of the invention, whenparticulate, will typically comprise particles with sizes ranging fromabout 1 nanometer to about 500 microns. In general, for solidformulations intended for intravenous administration, particles willtypically range from about 1 nm to about 10 microns in diameter.

The amount of the biologically active agent or drug in the formulationwill vary depending upon the specific drug employed, its molecularweight, and other factors such as dosage form, target patientpopulation, and other considerations, and will generally be readilydetermined by one skilled in the art. The amount of biologically activeagent in the copolymer formulation will be that amount necessary todeliver a therapeutically effective amount of the drug to a patient inneed thereof to achieve at least one of the therapeutic effectsassociated with the drug. In practice, this will vary widely dependingupon the particular drug, its activity, the severity of the condition tobe treated, the patient population, the stability of the formulation,and the like. Compositions will generally contain anywhere from about 1%by weight to about 30% by weight drug, typically from about 2% to about20% by weight drug, and more typically from about 3% to about 15% byweight drug, and will also depend upon the relative amounts ofexcipients/additives contained in the composition. More specifically,the composition will typically contain at least about one of thefollowing percentages of the entrapped drug: 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, or more by weight.

IV. Methods of Making the Block Copolymer

The multi-arm block copolymers of the invention can be prepared bysimply covalently attaching a preformed hydrophobic polymer segment tothe core molecule followed by covalently attaching a preformedhydrophilic polymer segment to the hydrophobic polymer segment.Alternatively, one or more of the polymer segments can be prepared bydirectly polymerizing monomer units of the polymer using, for example, aring-opening polymerization technique.

For example, in order to synthesize a poly(propyleneoxide)-poly(ethylene glycol) copolymer (PPO-PEG) on a polyol core, thepropylene oxide monomers can be directly polymerized onto the polyolcore by base-initiated ring-opening polymerization in a suitablesolvent. Suitable bases include potassium naphthalenide, sodium hydride,sodium or potassium alkoxides, or other strong bases. Suitable solventsinclude tetrahydrofuran, dioxane, or toluene. In a second step, theproduct of the first reaction is reacted with monomer units of ethyleneoxide using a base and solvent as described for the first reaction. Themolecular weight of the PPO polymer formed in the first step iscontrolled by the molar ratio of the propylene oxide to that of thepolyol. The molecular weight of the PEG polymer formed in the secondstep is controlled by the molar ratio of the ethylene oxide to that ofthe PPO polymer formed in the first step.

In those embodiments utilizing a poly(hydroxyester) hydrophobic polymersegment and a PEG hydrophilic polymer segment, it is preferable todirectly polymerize the hydroxyester monomer onto the core molecule(e.g. a polyol) to create the poly(hydroxyester) portion of thecopolymer, followed by covalent attachment of the PEG polymer to thedistal terminus of the poly(hydroxyester) segment.

V. Methods of Loading the Drug into the Multi-Arm Block Copolymer

There are several methods for entrapping a biologically active agent ordrug within the hydrophobic region of the block copolymers of theinvention. In a first method, the hydrophobic drug and the copolymer areco-dissolved in an organic solvent and then dried to form a solidproduct. The solid product is redissolved in aqueous solution andfiltered to remove insoluble particles prior to use. In a second method,the hydrophobic drug is suspended in an aqueous solution of thecopolymer and subjected to ultrasonication for several hours in order tointimately contact the drug molecules and the hydrophobic cores of thecopolymer structures. The solution is then filtered to remove insolubleparticles. In a third method, the hydrophobic drug and the polymer aremixed in solid form and heated to about 60° C. to form a melt. The meltis stirred for several hours to encourage intimate mixing of the drugand copolymer. After cooling to room temperature, the formulation isready for immediate use or storage.

VI. Method of Using the Multi-Arm Block Copolymers

As noted above, the multi-arm block copolymers of the invention can beused to solubilize hydrophobic drug molecules in aqueous solution. As aresult, the copolymer structures of the invention may be used as drugdelivery vehicles by entrapping the hydrophobic drug within thehydrophobic region of the copolymer and administering a therapeuticallyeffective amount of the multi-arm block copolymer with the biologicallyactive agent entrapped therein to a mammal.

The block copolymers of the invention can be used as drug deliveryvehicles for any condition responsive to a hydrophobic drug moleculecapable of entrapment within the copolymer structure. Thus, the blockcopolymers of the invention can be used in pharmaceutical formulationsuseful for treating any condition responsive to a hydrophobic drug inmammals, including humans. A preferred condition for treatment iscancer. The method of treatment comprises administering to the mammal atherapeutically effective amount of a composition or formulationcontaining the multi-arm block copolymer with a hydrophobic drugencapsulated therein as described above. The therapeutically effectivedosage amount of any specific formulation will vary somewhat from drugto drug, patient to patient, and will depend upon factors such as thecondition of the patient, the loading capacity of the block copolymer,and the route of delivery. As a general proposition, a dosage from about0.5 to about 20 mg/kg body weight, preferably from about 1.0 to about5.0 mg/kg, will have therapeutic efficacy. When administered conjointlywith other pharmaceutically active agents, even less of the blockcopolymer/hydrophilic drug composition may be therapeutically effective.

The block copolymer/hydrophilic drug composition may be administeredonce or several times a day. The duration of the treatment may be onceper day for a period of from two to three weeks and may continue for aperiod of months or even years. The daily dose can be administeredeither by a single dose in the form of an individual dosage unit orseveral smaller dosage units or by multiple administration of subdivideddosages at certain intervals. Possible routes of delivery includebuccally, subcutaneously, transdermally, intramuscularly, intravenously,orally, or by inhalation.

VII. Experimental

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention. Unless otherwiseindicated, all PEG reagents are available from Shearwater Corporation ofHuntsville, Ala. All NMR data was generated by a 300 MHz NMRspectrometer manufactured by Bruker.

Materials

Four 8-arm block copolymers were prepared. In each case, thepoly(propylene oxide) (PPO) segments of the copolymers were covalentlybonded to a hexaglycerol core through ether linkages and thepolyethylene glycol) (PEG) moiety was covalently bound to the distalterminus of each PPO segment. Copolymer PPO-PEG of nominal molecularweight 8500 Da was prepared with a 5300 Da PPO block and a 3200 Da PEGblock. Copolymer PPO-PEG 18000 (18000 Da molecular weight) was preparedwith a 5300 Da PPO block and a 12,700 Da PEG block. Copolymer PPO-PEG16000 was prepared with a 9500 Da PPO block and a 6500 Da PEG block.Copolymer PPO-PEG 22000 was prepared with a 9500 Da PPO block and a12,000 Da PEG block. It should be understood that these molecularweights are an average, nominal, molecular weight for polymers having arange of molecular weights. The general structure of a PPO-PEG copolymerof this type is given above as Formula V.

Additionally, a series of degradable multi-arm copolymers weresynthesized, in which degradable poly(hydroxyesters) were used ashydrophobic segments. These copolymers include 8-arm polylactidemPEG_(5kDa) (8-arm PLA-mPEG_(5kDa)), 8-arm polylactide PEG2_(6kDa)(8-arm-PLA-PEG2_(6kDa)), 8-arm polycaprolactone mPEG_(5kDa)(8-arm-PCL-mPEG_(5kDa)), 8-arm polycaprolactone PEG2_(6kDa)(8-arm-PCL-PEG2_(6kDa)), PEG2 attached to hydroxypropyl-β-cyclodextrinpolycaprolactone (BCD-PCL-PEG2_(6kDa)). All of the 8-arm degradablecopolymers were made using a hexaglycerol core. The general structure ofan 8-arm polylactide mPEG is given above as Formula VI. The 21-armBCD-PCL-PEG2_(6kDa) copolymer comprises a hydroxypropyl-β-cyclodextrincore. As used herein, PEG2 refers to a branched PEG structure comprisingtwo PEG backbones attached to a lysine linker, as described in U.S. Pat.No. 5,932,462. Examples 1-7 illustrate methods of synthesizing multi-armpoly(hydroxyester)-PEG copolymers.

For comparative purposes, the following additional materials weretested: Tetronic® 1037, a four arm PPO-PEG copolymer having nitrogenbranching points available from BASF Corp. (Mount Olive, N.J.); twomulti-arm PEG molecules available from NOF (Tokyo, Japan), a 4-atmpolymer comprising a pentaerythritol core and an 8-arm polymercomprising a hexaglycerol core; and Tween® 80, a polyoxyethylenesorbitan monooleate surfactant obtained from Aldrich (Milwaukee, Wis.).Physical data for all tested materials is listed in Table 1 below.

TABLE 1 Physical data of the materials used in the experiments PolymerMw (Da) Wt. % of PEG # of arm Non- PPO-PEG 6030 8500 36 8 degradablePPO-PEG 6070 18000 68 8 PPO-PEG 10037 16000 38 8 PPO-PEG 10050 2200054.5 8 Tetronic ® 1307 18000 70 4 PEG10kDa 10000 100 4 PEG10kDa 10000100 8 Tween 80 1310 67 N/A Degradable PLA-mPEG_(5kDa) 56000 71 8PLA-PEG2_(6kDa) 64000 75 8 PCL-mPEG_(5kDa) 56000 71 8 PCL-PEG2_(6kpa)64000 75 8 BCD-PCL- 168000 75 21 PEG2_(6kDa)

The following biologically active agents were used in the formulationand release studies detailed below: 3,4-di-[1-methyl6-nitro-3-indolyl]-1H-pyrrole-2,5-dione (MNIPD) (available from F.Hoffmann-La Roche Ltd, Basel, Switzerland), simvastatin (available fromMerck & Co., Inc., Whitehouse Station, N.J., USA), indomethacin(available from Sigma, St. Louis, Mo., USA), pivaloyloxymethyl butyrate(available from Titan Pharmaceuticals, Inc., San Francisco, Calif.,USA), cyclosporin A (available from Fluka, Milwaukee, Wis., USA), andpaclitaxel (available from LKT laboratories, Inc., St. Paul, Minn.,USA).

Drug Loading Methods

Three methods were used to load a hydrophobic drug into the multi-armblock copolymer formulations. Method I utilized an organic solvent.Method II utilized an aqueous solution. Method III was performed in theabsence of a solvent.

Method I: The hydrophobic drug and the copolymer were co-dissolved inmethylene chloride. The solution was air-dried overnight and then driedunder vacuum. The resulting solid was either stored at −20° C. forfuture use after thawing, dissolving in buffer, and filtering, or it wasdissolved immediately in a buffer, filtered to remove insolubleparticles and the filtrate frozen and stored at −20° C.

Method II: The hydrophobic drug was suspended in a buffered polymersolution. The suspension was subjected to ultrasonication for aboutthree hours, and then filtered through a 0.2 μm syringe filter. Thefiltrate was frozen and stored at −20° C.

Method III: The hydrophobic drug and the polymer were placed in a cappedvial under argon and heated to 60° C. to form a melt. The melt wasstirred for two hours using a magnetic stirrer. After cooling to roomtemperature, the formulation was ready for immediate use or storage forfuture use.

Example 1 Preparation of 8-Arm Polylactide-mPEG_(5kDa)(8-Arm-PLA-mPEG_(5kDa))

In a 250 mL three neck round bottom flask, hexaglycerol (4.307 gm, 0.008moles (Sakamoto Yakuhin Kogyo Co., Ltd., Osaka, Japan) was heated at100° C. for one hour under vacuum (1 mmHg). The contents were cooled toambient temperature and placed under argon. DL-lactide (160 gm, 1.110moles (Purasorb, Purac, Holland) was added and the flask flushed withargon then heated at 150° C. Stannous 2-ethyhexaneoate (94.6 mg,2.22×10⁻⁴ moles) was added and the mixture heated under argon at 170° C.for twenty-four hours. The mixture was cooled to 160° C. and stirredunder vacuum (less than 1 mm Hg) for three hours. After cooling to roomtemperature the mixture was dissolved in dichloromethane (900 mL). Thesolution was concentrated to near dryness at reduced pressure and pouredinto hexanes (1500 mL) with stirring to precipitate. The supernatant wasdecanted and the residue dried under vacuum. NMR (CDCl₃): δ 5.16 (m,—OCH(—CH₃)CO—), 1.57 (d, ill resolved, —OCH(—CH₃)CO—).

In a round-bottom flask, 8-arm PLA prepared from above (2 grams),mPEG_(5k)-CM (5 grams), 1-hydroxybenzotriazole (HOBT, 65 mg),4-(dimethylamino)pyridine (DMAP, 120 mg) and dicyclohexylcarbodiimide(DCC, 288 mg) were mixed with 40 ml of anhydrous methylene chloride. Themixture was stirred at room temperature overnight, the insoluble solidwas removed by filtration, and the solvent was evaporated under reducedpressure. The residue was added to 100 ml of ether and the resultingprecipitate was collected by filtration and dried under vacuum. Yield:5.5 g (78%). ¹H NMR (DMSO-d₆): δ 3.5 (br m, PEG), 4.20 (s,—PEG-OCH2COO-PLA), 5.16 (m, —OCH(—CH₃)CO—), 1.45 (d, ill resolved,—OCH(—CH₃)CO—).

Example 2 Preparation of 8-Arm Polylactide PEG2_(6kDa)(8-Arm-PLA-PEG2_(6kDa))

8-arm-polylactide (8-arm-PLA) (3.00 g, Mw ˜20 kDa), branched PEGcarboxylic acid (PEG2-COOH, 6 kDa, 7 g), DMAP (120 mg), HOBT (105 mg)and DCC (440 mg) were dissolved in methylene chloride (50 ml). Thereaction was stirred at room temperature for about 72 hours. The solventwas then removed under vacuum, and 35 ml of 1,4 dioxane was added to thesyrup. After filtering, the filtrate was added to 200 ml of diethylether. The precipitate was collected by filtration, washed withisopropyl alcohol (IPA) and ether, and then dried overnight undervacuum. Yield: 9.4 g. ¹H NMR (DMSO-d₆): δ 3.5 (br m, PEG), 1.45 (d,—OCCH(CH ₃)O—), 5.165 (m, OCCH(CH₃)O—), 4.03 (t, mPEGOCH₂CH ₂OCONH—).

Example 3 Preparation of 8-Arm ε-Polycaprolactone (8-Arm PCL)

Hexaglycerol (2.156 g) was dried by heating at 100° C. for 16 hoursunder vacuum. Five ml of N,N-dimethyl formamide was added and themixture heated under argon to 80° C. To the resulting mixture was added80 g (74 ml) of ε-caprolactone (Aldrich) and stannus ethyl hexaneoate(48 mg). The mixture was heated to 110° C. for ˜72 h. The flask wascooled and unreacted reagent and solvent were removed under vacuum.Yield: ˜80 g. ¹H NMR (DMSO-d₆): 1.23 (br m, —OCCH₂CH₂CH ₂CH₂CH₂O—), 1.52(m, —OCCH₂CH ₂CH₂CH ₂CH₂O—), 2.26 (t, —OCCH ₂CH₂CH₂CH₂CH₂O—), 3.98 (t,—OCCH₂CH₂CH₂CH₂CH ₂O—). The molecular weight of the 8-arm-PCL wasestimated as 16000 Dalton by NMR and GPC.

Example 4 Preparation of 8-Arm Polycaprolactone mPEG_(5kDa)(8-Arm-PCL-mPEG_(5kDa))

8-arm-PCL from Example 3 (1.00 g), carboxymethyl mPEG_(5kDa) (2.10 g),DMAP (60 mg), HOBT (35 mg) and DCC (140 mg) were dissolved in methylenechloride (30 ml). The reaction was stirred at room temperature for 46hours. The solvent was then removed under vacuum, and 15 ml of 1,4dioxane was added to the syrup. After filtering, the filtrate wasconcentrated by removing excess 1,4 dioxane under vacuum. The productwas precipitated with 200 ml of diethyl ether, stirred for 5 minutes,and collected by filtration. The product was dried overnight undervacuum. Yield: 2.6 g. ¹H NMR (DMSO-d₆): δ 3.5 (br m, PEG), 4.20 (s,-PEG-OCH ₂COO-PCL), 1.28 (br m, —OCCH₂CH₂CH ₂CH₂CH₂O—), 1.55 (m,—OCCH₂CH ₂CH₂CH ₂CH₂O—), 2.26 (t, —OCCH ₂CH₂CH₂CH₂CH₂O—), 3.99 (t,—OCCH₂CH₂CH₂CH₂CH ₂O—).

Example 5 Preparation of 8-Arm Polycaprolactone PEG2_(6kDa)(8-arm-PCL-PEG2_(6kDa))

8-arm-PCL (1.00 g), branched PEG carboxylic acid (PEG2_(6kDa)-COOH, 2.52g), DMAP (60 mg), HOBT (35 mg) and DCC (140 mg) were dissolved inmethylene chloride (30 ml). The reaction was stirred at room temperaturefor about 72 hours. The solvent was then removed under vacuum, and 15 mlof 1,4 dioxane was added to the syrup. After filtering with celite, thefiltrate was concentrated by removing excess 1,4 dioxane under vacuum.The product was precipitated with 200 ml of diethyl ether, stirred for 5minutes, collected by filtration, and dried overnight under vacuum.Yield: 3.1 g ¹H NMR (DMSO-d₆): δ 3.5 (br m, PEG), 1.28 (br m,—OCCH₂CH₂CH₂CH ₂CH₂O—), 1.55 (m, —OCCH₂CH ₂CH₂CH ₂CH₂O—), 2.26 (t, —OCCH₂CH₂CH₂CH₂CH₂O—), 3.99 (t, —OCCH₂CH₂CH₂CH₂CH ₂O—).

Example 6 Preparation of Polycaprolactone Initiated withHydroxypropyl-β-cyclodextrin (BCD-PCL)

Hydroxypropyl-β-cyclodextrin (BCD; 100 substitution) was purchased fromAldrich and used as received. ε-Caprolactone (CL; Aldrich) was purifiedby dehydration with CaH₂ and distillation under vacuum. The purifiedproduct was stored under N₂ atmosphere at −20° C. until use. Stannous2-ethyhexaneoate (SnOct; Aldrich) and all other reagents were used asreceived.

Hydroxypropyl-β-cyclodextrin (BCD, 1.45 gram, 1 mmol) was vacuum-driedin a round-bottomed flask at 100° C. for 1 hour and purged with dry N₂.Purified ε-caprolactone (42 gram, 0.368 mol) was added to the flaskusing a syringe. Thirty-two milligrams of stannous 2-ethyhexaneoate(SnOct, Aldrich) was added, and the mixture stirred for 24 hours. Themixture of reagents became viscous without significant change in color.While the mixture was cooled, tetrahydrofuran (100 ml) was added. Thepolymer was precipitated by addition of about 2 L of isopropanol (IPA).The precipitate was collected by filtration and redissolved in benzeneand freeze-dried for 2 days. Yield: 37 g (86%).

Example 7 Preparation of PEG2 Attached to BCD-PCL (BCD-PCL-PEG2_(6kDa))

One gram of BCD-PCL was mixed with 3.8 g (0.025 mmol) of PEG2-carboxylicacid (MW6,000), 0.866 g of dicyclohexyl carbodiimide (4.2 mmol), 0.122 gof 4-dimethylaminopyridine (1.0 mmol) and 0.068 g ofhydroxybenzotriazole (0.5 mmol) in 20 ml of 1,2-dichloroethane (ordichloromethane), and stirred for 48 hours. The solvent was removedunder vacuum, and the remaining gummy material gummy material wasdissolved in 40 ml of 1,4-dioxane. The undissolved material was removedby filtration and the solution was added to 400 ml of diethylether. Theprecipitate was filtered and dried under vacuum for 48 h. Yield: 4.2gram (88%).

Example 8 Synthesis of Bisphosphonate Derivative of Multi-Arm PPO-PEG

8 Arm PPO-PEG (18 KDa) (succinimidyl carbonate)₈:

8 arm PPO-PEG (18 KDa) (15.0 g, ˜0.83 mmol) in acetonitrile (200 mL) wastreated with disuccinimidyl carbonate (DSC) (1.9 g, 7.4 mmol) andpyridine (0.70 ml). The reaction was stirred overnight at roomtemperature under an argon atmosphere. The reaction was concentrated todryness and the residue was dissolved in dichloromethane (˜200 ml). Theclear solution was washed with a 10% solution of sodium phosphate,sodium chloride (2×200 ml). The organic layer was dried (Na₂SO₄),filtered, and the solvent was removed to afford 8 arm PPO-PEG (18KDa)-(a-succinimidyl carbonate)₈ (15.0 g, ˜100%).

8 Arm PPO-PEG (18 KDa) (AHBDP)₅:

8 arm PPO-PEG (18 KDa)-(α-succinimidyl carbonate)₈ (10.0 g, ˜0.55 mmol)and 4-amino-1-hydroxybutane-1,1-diphosphonic acid, ditetrabutylammoniumsalt (AHBDP) (2.96 g, 3.76 mmol) were dissolved in acetonitrile (200 ml)and treated with triethylamine (0.8 ml, 5.74 mmol). The clear, colorlesssolution was stirred overnight under and argon atmosphere. The solutionwas concentrated to dryness, the residual gum dissolved in water (100mL), and the pH was adjusted to 11. The basic solution was stirred atroom temperature for 2 h. The solution was then adjusted pH 7.0 with HCland passed through an IR 120 column (75 ml). The water was removed invacuo at ca. 50° C. to afford the product as a gum. Further drying invacuo followed by trituration with CH₂Cl₂ with Et₂O afforded the productas a waxy solid (4.5 g). ¹H NMR (dmso-d₆, 300 MHz) δ 1.04 (d, 280H,OCH(CH₃)CH₂), 1.59-1.76 (m, 12.5H, OCONHCH₂CH₂CH₂), 1.76-1.94 (m, 12.4H,OCONHCH₂CH₂CH₂), 2.88-2.99 (m, 12.9H, OCONHCH₂CH₂CH₂), 3.51 (bs, 1191H,PEG backbone), 4.03 (t, 13H, J=4.4 Hz, CH₂CH₂OCONH), 7.16 (t, 5.0H,J=5.1 Hz, CH₂CH₂OCONH).

Example 8 illustrates a method of synthesizing a multi-arm copolymerincluding a targeting moiety attached to a distal end of the outer PEGpolymer.

Example 9 Preparation of Cyclosporin A-Loaded 8-Arm-PCL-PEG2_(6kDa)

In a glass vial, 6 mg of cyclosporin A and 60 milligram of8-arm-PCL-PEG2_(6kDa) (drug/polymer weight ratio 1/10) were dissolved in1 ml of methylene chloride. The solution was dried under argon. Thedried solid was heated at 55° C. for two hours under argon. The melt wasthen cooled to room temperature, placed under vacuum overnight, andreduced to small particles. To the particles was added 1 ml of phosphatebuffer (0.1 M, pH 7.0), and the resulting mixture was filtered through0.2 μm syringe filter. Cyclosporin A concentration was 5.5 mg/ml byHPLC.

Example 10 Preparation of Paclitaxel-Loaded 8-Arm-PCL-PEG2_(6kDa)

Paclitaxel (6 mg) and 8-arm-PCL-PEG2_(6kDa) (60 mg) (drug/polymer weightratio 1/10) were dissolved in 1 ml of methylene chloride. The solutionwas dried under argon. The dried solid was heated at 55° C. for twohours under argon. The melt was then cooled to room temperature andplaced under vacuum overnight, and reduced to small particles. To theparticles was added 1 ml of phosphate buffer (0.1 M, pH 7.0). Theresulting mixture was filtered through 0.2 μM syringe filter. Paclitaxelconcentration was above 4.5 mg/ml by HPLC.

Example 11 Solubility of Drugs in PPO-PEG Multi-Arm Block Copolymers

For several drug molecules, 50 mg of PPO-PEG block copolymer 10050/drugformulation with 10 wt. % of drug loading was dissolved in 1 ml ofphosphate buffer (0.1 M, pH 7.4). After two hours of mixing, the mixturewas filtered through 0.2 μm syringe filter. The drug concentration inthe filtrate was determined by HPLC or UV using a standard curve. Theresults are listed in Table 2. As noted in Table 2, in each case,incorporation of the drug into the multi-arm PPO-PEG block copolymergreatly increased the solubility of the drug in buffer solution.

TABLE 2 Solubility of Drug in 50 mg of the Multi-Arm PPO-PEG BlockCopolymer (phosphate buffer, 0.1 M, pH 7.4) Pivaloyloxymethyl Drug MNIPDSimvastatin Indomethacin Paclitaxel butyrate Solubility of Drug in <0.5μg/ml <1 μg/ml 88 μg/ml <1 μg/ml — Plain Buffer Solubility of Drug in  2.6 mg/ml   4 mg/ml  4 mg/ml   2 mg/ml 12 mg /ml Copolymer/DrugFormulation Solubility of ~5,000 ~4,000 ~45 ~2,000 — FormulationRelative to Solubility in Buffer

Example 12 Degradation Studies of Degradable Multi-Arm Block Copolymers

Each of the multi-arm PEG block copolymers having degradable hydrophobicsegments was dissolved in either phosphate buffer (0.1 M, pH 7.0) or ratserum to a final concentration of 1-4 wt. %. The solution was placed inan incubator at 37° C. The concentrations of the copolymer and free PEGwere monitored at timed intervals by HPLC. For the solution in ratserum, the copolymer and PEG were first extracted with methylenechloride and then analyzed by HPLC, while for the solution in buffer,analysis was done directly by HPLC. Half-lives (t½) were calculatedbased on first order kinetics, as shown in Table 3. The data indicatethat all of the tested polymers are degradable in both rat serum andphosphate buffer with varying degradation rates depending on thestructure of the polymer. Larger PEG segments tended to result in longerdegradation half-lives.

TABLE 3 Degradation Half-Lives (t1/2) of Selected Multi-Arm BlockCopolymers Sample In phosphate buffer (pH 7.0) In rat serum8-arm-PLA-PEG5k  365 h  7 h 8-arm-PLA-PEG2_(6kDa) 1251 h  8 h8-arm-PCL-PEG5k  643 d 170 h 8-arm-PCL-PEG2_(6kDa) 1010 d 507 h

Example 13 Drug Release Studies Using PPO-PEG Multi-Arm Block Copolymers

In aqueous media, complexes of soluble lipophilic/hydrophobic drugs withthe multi-arm PPO-PEG copolymer slowly release the drug. Thewater-insoluble drugs precipitate out of the formulation over time.Release profiles of the drugs were studied by determining theconcentration of solubilized drug as a function of time at 23° C. Ateach time interval, aliquots were filtered through a 0.2 μm syringefilter and concentrations of drug measured by rp-HPLC or UV methods. Forexample, release of MNIPD from the soluble MNIPD/PPO-PEG formulationwere measured by withdrawing 100 μl of solution, diluting to 1000 μl inwater (at which point the drug dissolves), filtering through a 0.2 μmfilter, and measuring absorbance of the filtrate at 465 nm. Drug releasecurves are presented in FIGS. 2-7.

In FIG. 2 is shown a comparison of the release rate of the drug, MNIPD,from PPO-PEG multi-arm copolymers 6035 and 6070 with the release rate ofMNIPD from multi-arm PEGs (4-arm and 8-arm) and from Tween 80 (see Table1). MNIPD was loaded into the polymers by Method I. After theMNIPD/polymer formulation was dissolved in phosphate buffer (0.1 M, pH7), the release of the drug was followed by UV at 465 nm. The drug wasreleased more slowly from copolymers 6035 and 6070 than from Tween 80.Solubility in the 4- and 8-arm PEGs was low and the drug was rapidlyreleased from these polymers.

Release profiles of simvastatin from seven polymers are shown in FIG. 3.Simvastatin was loaded into the polymers by Method I. After dissolvingin phosphate buffer (0.1 M, pH 7), the release of the drug was followedby HPLC. An extended release profile was observed from block copolymers10050 and 10037 (see Table 1), while release from the PEGs (4-arm and8-arm), 1307 and PPO-PEG 6035 was significantly more rapid. Solubilityin the multi-arm PEG molecules and in PPO-PEG 6070 was very low.

In FIG. 4 is shown a release profile for simvastatin from PPO-PEGcopolymer 10050 having a bisphosphonate targeting group attached to adistal terminus of the PEG moiety of the copolymer. Simvastatin wasloaded into the bisphosphonate derivative of the copolymer by method I.After the drug/copolymer formulation dissolved in phosphate buffer (0.1M, pH 7), the release of the drug was followed by HPLC. The drug wasreleased over about 80 hours.

In FIG. 5 is shown a comparison of release profiles of paclitaxel fromcopolymer 10050 and from 8-arm PLA-PEG block copolymer. Paclitaxel wasloaded into the copolymers by method I. After dissolving in phosphatebuffer (0.1 M, pH 7), the release of the drug was followed by HPLC.Higher drug loading was possible with the 8-arm PLA-PEG copolymer.

In FIG. 6 is shown a comparison of release profiles of indomethacin fromvarious copolymers. Indomethacin was loaded into the copolymers byMethod I. After dissolving in phosphate buffer (0.1 M, pH 7), therelease of the drug was followed by HPLC. Drug solubility was enhancedby the multi-arm block copolymers as well as by multi-arm PEG. Little orno release was observed from any of the polymers.

In FIG. 7 is shown comparative release profiles for pivaloyloxymethylbutyrate at two concentrations in PPO-PEG copolymers 10050 and 10037.Pivaloxymethyl butyrate was loaded into the copolymers by Method III.After dissolving in phosphate buffer (0.1 M, pH 7), the release of thedrug was followed by HPLC. Extended release was observed from bothpolymers.

Example 14 Release Profile of Cyclosporin A from Degradable8-arm-PCL-PEG2_(6kDa)

The solution prepared in Example 9 was incubated at 37° C. At timedintervals, 10 μl of sample was withdrawn and diluted with phosphatebuffer (0.1 M, pH 7.0). The solution was filtered through 0.2 μm syringefilter. The filtrate was analyzed by rp-HPLC for cyclosporin Aconcentration. The soluble cyclosporin A concentration in solution vs.time is shown in FIG. 8. The data illustrate the ability of the blockcopolymer to retain the cyclosporin A in solution for an extended periodof time and to provide a controlled release of the drug.

Example 15 Release Profile of Paclitaxel from Degradable8-Arm-PCL-PEG2_(6kDa)

The solution prepared in Example 10 was incubated at 37° C. At timedintervals, 10 μl of sample was withdrawn and diluted with phosphatebuffer (0.1 M, pH 7.0). The solution was filtered through 0.2 μM syringefilter. The filtrate was analyzed by rp-HPLC for paclitaxelconcentration. The soluble paclitaxel concentration in solution vs. timeis shown in FIG. 9. The data illustrate the ability of the blockcopolymer to retain the paclitaxel in solution for an extended period oftime and to provide a controlled release of the drug.

Example 16 Antitumor Study of Paclitaxel-Loaded 8-Arm PLA-mPEG_(5kDa) inNCI-H460 Non-Small Cell Lung Tumor Xenograft in Mice

NCI-H460 non-small cell lung tumor was implanted subcutaneously inathymic nude mice. After the tumor grew to approximately 175 mg, theaqueous formulation of paclitaxel-loaded 8-arm PLA-mPEG_(5kDa) wasinjected into mice via the tail vein. The mice were observed daily forsurvival. Tumor weights and body weights were recorded twice weekly.Each tumor was measured by caliper in two dimensions and converted totumor mass using the formula for a prolate ellipsoid. For comparison, astandard formulation of Taxol® (in Cremophor®) and a control were alsoused. The results are shown in FIG. 10. The data indicate that theinhibition of tumor growth exhibited by the 8-arm PLA-mPEG blockcopolymer/drug formulation (referred to as UM-Paclitaxel in FIG. 10) wascomparable to the standard Taxol® formulation.

Example 17 Tolerance Study of Multi-Arm Block Copolymers in Mice

Dosages ranging from 500 to 2000 mg/kg/dose were intravenouslyadministered to athymic nude mice for five days (days 1-5). As indicatedin Table 4, all dosages were well tolerated.

TABLE 4 Tolerance study of multi-arm copolymers Unimolecular micelleMean animal weight polymers loss during 21 days 21-day survival8-arm-PLA-PEG5k <5% All 8-arm-PLA-PEG₂6k <5% All 8-arm-PCL-PEG₂6k <5%All

Example 18 Dynamic Light Scattering Study

Micelle Preparation:

A block copolymer (0.14 g) selected from the group including linearPEG-PLA, 8-arm-PLA-PEG5k, 8-arm-PLA-PEG2_(6kDa), 8-arm-PCL-PEG5k, and8-arm-PCL-PEG2_(6kDa) was dissolved in 20 ml of N-dimethylacetamide(DMAc). The solution was warmed in order to dissolve the polymer easily.The solution was put into the pre-swollen dialysis membrane(Spectra/Prol, MWCO 6000-8000) after 0.2 μm filtration. Dialysis wascarried out against deionized water for 24 h. Water was changed at 1, 2,4 and 7 hours from the beginning. The prepared micelle solution wasstored at 4° C. until use.

Micelles Loaded with Paclitaxel:

Taxol® was loaded into the micelle solution in two ways. Method 1: Tothe micelle solution (10 ml) prepared as described above was added 0.5ml of paclitaxel solution in CHCl₃ (4 mg/mL) dropwisely. After 16 h ofvigorous stirring, CHCl₃ was removed from the solution by aspiration.The solution was filtered through 0.2 μm membrane. Method 2: Blockcopolymer (0.14 g) and paclitaxel (5 mg) were dissolved in DMAc anddialyzed as described above. After dialysis, the solution was filteredwith 0.2 μm membrane.

Dynamic Light Scattering (DLS):

The size and the distribution of micelles were measured by dynamic lightscattering. The sample was filtered with 0.2 μm pore-size membrane priorto the measurement. The measurement was carried out at 25° C. Size andpolydispersity of the particle was determined by cumulant analysismethod based on the assumption that the micelles were spherical. FIG. 11provides an example of dynamic light scattering results. Tables 5, 6 and7 provide micelle sizes and polydispersity for micelles with and withoutpaclitaxel loading as determined by light scattering.

TABLE 5 Size of Micelles Determined by Light Scattering Effective SampleDiameter (nm) Polydispersity Count Rate Linear PEG-PLA 30.4 0.198 313.88-arm-PLA-PEG5k 47.0 0.291 123.5 8-arm-PLA-PEG2_(6kDa) 100.7 0.351 251.98-arm-PCL-PEG5k 24.9 0.066 101.2 8-arm-PCL-PEG2_(6kDa) 19.3 0.079 73.3

TABLE 6 Size of Micelles Loaded with Paclitaxel Prepared by Method 1Effective Sample Diameter (nm) Polydispersity Count Rate Linear PEG-PLA39.1 0.226 363.0 8-arm-PLA-PEG5k 8-arm-PLA-PEG2_(6kDa) 8-arm-PCL-PEG5k49.0 0.106 692.0 8-arm-PCL-PEG2_(6kDa) 26.2 0.177 153.3

TABLE 7 Size of Micelles Loaded with Paclitaxel Prepared by Method 2Effective Sample Diameter (nm) Polydispersity Count Rate Linear PEG-PLA31.8 0.246 312.3 8-arm-PLA-PEG5k 8-arm-PLA-PEG2_(6kDa) 86.4 0.342 235.78-arm-PCL-PEG5k 8-arm-PCL-PEG2_(6kDa) 19.7 0.064 77.3

The above data indicate that the effective diameter of the multi-armblock copolymer structure increases after loading with the drug.

Example 19 Evaluation of Micelle Aggregates by Dynamic Light Scattering

Micelle solutions of BCD-PCL-PEG2_(6kDa), 8-arm-PCL-PEG2_(6kDa), andlinear PEG-PCL (MW 5,000-5,000) were prepared by a dialysis method. Forlinear PEG-PLA, the polymer solution in DMAc was mixed with water bydropwise adding 20 mL of water to the polymer solution prior to thedialysis in order to avoid the formation of aggregation. Theconcentration of micelle solutions was in the range of 2.88-3.34 mg/ml(see Table 8).

The micelle solutions were treated with ˜2.5 ml of 5% SDS solution for24 hours. The micelle solutions before and after the addition ofsurfactant (SDS) were characterized after filtration through 0.2 μmsyringe filter using a Brookhaven 90 Plus Particle Sizer. Table 4summarizes the DLS cumulant analysis results. The cumulant diameter ofthe micelles ranged from 19 to 35 nm before the SDS addition. The DLSmeasurement was also carried out without filtration, and littlealteration of micelle property was seen on the multi-arm blockcopolymers. BCD-PCL-PEG2_(6kDa) had the least change in micelle propertybefore and after SDS addition. The other micelles presented dramaticchange in size and significant decrease in count rate. The count rate ofthe BCD-PCL-PEG2_(6kDa) micelles was reduced by 30%. This was likely dueto the dilution of the micelle solution rather than the dissociation ofmicelles. Multi-armed PEG block copolymers with higher number of armstend to be less aggregated.

The data tends to suggest that increases in the number of arms of themulti-arm block copolymers of the present invention reduces the tendencyof the hydrophobic cores of the copolymers to aggregate in the samemanner as conventional linear micelles. Since less aggregation occurswith multi-arm copolymers with a greater number of arms, lessdisaggregation is caused by addition of the surfactant. In contrast,smaller block copolymers of the invention and linear micelles tend toaggregate to a greater extent, thereby resulting in a measurabledisruption in aggregation by the surfactant.

TABLE 8 DLS cumulant analysis results of BCD-PCL--(PEG3k)₂,8-arm-PCL-PEG2_(6kDa), and linear PEG-PCL micelles Concentration Countrate % Count Samples (mg/mL) Diameter (nm) Dispersity (kcps) RemainingBCD-PCL-PEG2_(6kDa 2) 3.34 19.8 0.098 52.7 Before filterBCD-PCL-PEG2_(6kDa) 3.34 19.4 0.058 44.2 After filterBCD-PCL-PEG2_(6kDa) + 3.34 22.7 0.152 30.7 69.45701357 SDS8-arm-PCL-PEG2_(6kDa) 3.24 Before filter 8-arm-PCL-PEG2_(6kDa) 3.24 26.80.158 107.1 After filter 8-arm-PCL-PEG2_(6kDa) + 3.24 42.2 0.24 23.521.94211018 SDS Linear PEG-PCL 2.88 Before filter Linear PEG-PCL 2.8835.6 0.006 348.7 After filter Linear PEG-PCL + SDS 2.88 11 0.401 113.154574132

Example 20 PEG Based Unimolecular Micelle Delivery Systems: Compositionand Characterization

Multi-arm polyethylene glycol-based unimolecular micelles weresynthesized using polycaprolactone (PCL) or polylactic cid (PLA)segments as the core of each micelle. Each arm (2 kDa) of the core wasconjugated to a linear or branched PEG chain (5 or 6 kDas). Thesolubility of paclitaxel in the PEG-PCL and PEG-PLA polymers wascompared with that of a water soluble cyclodextrin (HP-β-CD). The drugwas then incorporated into a branched PEG6 kDa-PCL polymer using anorganic complexation technique and then formulated with mannitol toprepare water soluble lyophilized formulations. Several formulation lotswere placed on a 6-month accelerated stability study and assayed atregular intervals for drug content, polymer molecular weight, organicvolatile impurities, moisture, pH, osmolality and clarity uponreconstitution.

PEG-based unimolecular micelles provide at least a 40-fold improvementin solubility when compared to HP-(3-CD. The branched PEG6 kDa-PCLpaclitaxel formulation prepared was a water soluble white porous cakethat, upon reconstitution, remained clear for at least 24 hours at aconcentration of 3.6 mg/mL of paclitaxel. It was found to be stableduring the 6 month testing period when stored at 2-8° C., 25° C./60% RHand 40° C./75% RH. The specifications for this product are shown inTable 9, below:

TABLE 9 Specifications For Branched PEG6kDa-PCL Paclitaxel FormulationPolymer OVI Drug molecular (ppm of Appearance Osmolality concentrationweight methylene and Clarity pH (mOsm/Kg) (mg/mL) (Daltons) Moisturechloride) White porous 7.20 300 3.6 55,000 <1.0% <50 cake, clear uponreconstitution

Based on the results described herein, it is possible to conclude thataqueous PEG based unimolecular micelles are good solubilizing agents forhighly water insoluble compounds. A novel unimolecular micelleformulation of paclitaxel was developed which provided adequate drugloading and good stability.

Example 21 Bioequivalence of Aqueous PEG-Based Unimolecular MicelleFormulations of Cyclosporin and Tacrolimus

The oral (PO) bioavailability of aqueous PEG-based unimolecular micelle(UM) formulations of cyclosporine and tacrolimus in rats was determined.

Multi-arm polyethylene glycol-based unimolecular micelles (64 kDas) weresynthesized using polycaprolactone (PCL) segments conjugated to abranched PEG chain. cyclosporin A (CSA) and tacrolimus (TAC) wereseparately incorporated into the PEG-PCL polymer using an organiccomplexation technique and then formulated with mannitol to preparewater soluble lyophilized formulations. Studies in rats compared thebioavailability of CSA or TAC unimolecular micelle preparations againstmarketed comparator formulations. Blood samples were collected over 24hours from each animal through an in-dwelling jugular catheter, withdonor blood re-infused at each collection. Concentrations of CSA or TACin rat plasma were quantified using an LC/MS/MS method. The method hadan assay range of 1-3000 ng/mL in plasma.

Each formulation studied was tolerated with no adverse effects reported.A summary of the pharmacokinetic analysis (WINNONLIN, non-compartmental)is provided in Table 10 below (“IV” represents intraveneous):

TABLE 10 Summary of Pharmacokinetic (PK) Analysis of Tested FormulationsCSA-UM CSA-UM Neoral ® TAC-UM TAC-UM Prograf ® PK IV 5 PO 20 PO 20 IV 1PO 4 PO 4 parameter mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Cmax NA 1.60 ±0.28 2.62 ± 0.26 NA 0.17 ± 0.03 0.12 ± 0.03 (μg/mL) Tmax NA 2.67 ± 1.163.33 ± 2.31 NA 0.50 ± 0.00 1.17 ± 0.76 (h) t½ 9.90 ± 1.50 9.40 ± 2.0011.60 ± 0.30  7.40 ± 0.40 9.50 ± 3.10 7.60 ± 2.00 (h) CL 0.33 ± 0.01 NANA 3.14 ± 0.22 NA NA (L/h/Kg) Vss 4.05 ± 0.46 NA NA 20.75 ± 1.56  NA NA(L/Kg) F NA 41.69 71.82 NA 36.15 28.86 (%)

Based on the results described herein, it is possible to conclude thatstable PEG-based aqueous parenteral formulations were developed. TheUM-TAC oral formulation was bioequivalent when compared to the marketedPrograf® product. UM-CSA was found to be less bioavailable compared toNeoral®.

Example 22 Efficacy and Distribution of Paclitaxel in Murine XenograftModel when formulated in Aqueous PEG Based Unimolecular Micelles

The in vivo anti-tumor activity and pharmacokinetics of a novelunimolecular micelle formulation of paclitaxel (UM-paclitaxel) in ahuman NSCLC NCI-H460 murine xenograft model were assessed.

The in vivo anti-tumor activity of UM-paclitaxel (30, 20, 10 mg/kg) wasevaluated against the human NSCLC NCI-H460 xenograft tumor in nude(nu/nu) athymic mice. Paclitaxel (10 mg/kg, the maximum tolerated dosein this model) and vehicle were used as positive and negative controls.Following tumor establishment, mice were allocated to treatment groups,treated every day for 5 days and followed for tumor progression. Aseparate pharmacokinetic (PK) study with a similar dosing schedulecompared serum and tumor paclitaxel concentrations of the differentformulations (UM-paclitaxel 20, 10 mg/kg; paclitaxel 10 mg/kg).

Treatment with UM-paclitaxel resulted in a dose-related reduction intumor growth at all dose levels tested throughout the duration of thestudy. The reduction in mean relative tumor volume was statisticallysignificant at 30 mg/kg for the duration of the study. The novelpaclitaxel formulation was well-tolerated, with some reversibledeterioration in skin condition and mild weight-loss noted in the 30mg/kg group. Paclitaxel-treated animals displayed transient prostration5-10 minutes post-dosing, but was otherwise well-tolerated. In the PKstudy, serum concentration-time profiles of animals treated with 10mg/kg were similar between the two formulations. UM-paclitaxel dosed at20 mg/kg produced higher serum concentrations, a longer half-life anddisproportionate increases in serum AUC. Tumor paclitaxel concentrationsin the 20 mg/kg UM-paclitaxel-treated animals increased over the 5-daydosing period, suggesting an accumulation of drug. This was not observedwith either formulation at the lower doses studied.

Based on the results described herein, it is possible to conclude thatthe UM-paclitaxel formulation was generally well-tolerated.Pharmacokinetic analysis suggested accumulation within the tumor duringthe dosing period studied.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andthe associated tables. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

What is claimed:
 1. A multi-arm block copolymer encompassed by thefollowing formula:

wherein each PEG is a poly(ethylene oxide) and each PPO is apoly(propylene oxide).